The pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection involves dysregulations of iron metabolism, and although the mechanism of this pathology is not yet fully ...understood, correction of iron metabolism pathways seems a promising pharmacological target. The previously observed effect of inhibiting SARS-CoV-2 infection by ferristatin II, an inducer of transferrin receptor 1 (TfR1) degradation, prompted the study of competition between Spike protein and TfR1 ligands, especially lactoferrin (Lf) and transferrin (Tf). We hypothesized molecular mimicry of Spike protein as cross-reactivity of Spike-specific antibodies with Tf and Lf. Thus, strong positive correlations (R
2
> 0.95) were found between the level of Spike-specific IgG antibodies present in serum samples of COVID-19-recovered and Sputnik V-vaccinated individuals and their Tf-binding activity assayed with peroxidase-labeled anti-Tf. In addition, we observed cross-reactivity of Lf-specific murine monoclonal antibody (mAb) towards the SARS-CoV-2 Spike protein. On the other hand, the interaction of mAbs produced to the receptor-binding domain (RBD) of the Spike protein with recombinant RBD protein was disrupted by Tf, Lf, soluble TfR1, anti-TfR1 aptamer, as well as by peptides RGD and GHAIYPRH. Furthermore, direct interaction of RBD protein with Lf, but not Tf, was observed, with affinity of binding estimated by K
D
to be 23 nM and 16 nM for apo-Lf and holo-Lf, respectively. Treatment of Vero E6 cells with apo-Lf and holo-Lf (1–4 mg/mL) significantly inhibited SARS-CoV-2 replication of both Wuhan and Delta lineages. Protective effects of Lf on different arms of SARS-CoV-2-induced pathogenesis and possible consequences of cross-reactivity of Spike-specific antibodies are discussed.
•Asymmetry of DNA methylation of sister chromatids decreased upon BPA action.•Preimplantation embryos and F9 cells are most sensitive targets to BPA influence.•Nuclei methylation decreased in ...preimplantation embryos upon BPA action.
Earlier we showed that asymmetric methylation of sister chromatids (AMSC) was a specific characteristic of differentiation potency, and supposed that AMSC could be a useful marker of environmental impact connected with differentiation and/or dedifferentiation. Here we investigated the level of AMSC in chromosomes and the nuclei methylation in mouse preimplantation and postimplantation embryos, in comparison with the undifferentiated cells of mouse embryonal carcinoma cell line F9, and human differentiated HEK293 cells upon BPA influence. We found that exposure of mouse preimplantation embryos to BPA caused a significant decrease in the level of AMSC in chromosomes and the nuclei methylation. The BPA exposure of potentially differentiating F9 cells had no any influence on DNA methylation in nuclei but significantly decreased the number of AMSC. The level of DNA methylation and AMSC in HEK293 cells were not also changed. These data indicate that BPA exerts significant influence on differentiating and potentially differentiable cells. The most sensitive BPA targets are preimplantation embryos and stem cells.
Macrophage migration inhibitory factor (MIF) is a key proinflammatory cytokine. Inhibitors of tautomerase activity of MIF are perspective antiinflammatory compounds. Ceruloplasmin, the ...copper-containing ferroxidase of blood plasma, is a noncompetitive inhibitor of tautomerase activity of MIF in the reaction with
p
-hydroxyphenylpyruvate. Small-angle X-ray scattering established a model of the complex formed by MIF and ceruloplasmin. Crystallographic analysis of MIF with a modified active site supports the model. The stoichiometry of 3 CP/MIF trimer complex was established using gel filtration. Conformity of novel data concerning the interaction regions in the studied proteins with previous biochemical data is discussed.
This manuscript describes the chemical synthesis of a compound similar to fluorene and Congo red, including characterization of its spectral properties. It was shown that the dye, during interaction ...with amyloid-like fibrils of several proteins (lysozyme, insulin, and beta-2-microglobulin), has the ability to change fluorescent spectrum. In contrast, monomeric forms of these proteins did not induce significant spectral changes.
•The AF dye changes its spectral characteristics when bound to amyloid-like fibrils.•The amyloid detecting properties were verified using three amyloidogenic proteins.•Slight changes in AF dye spectra were observed when binding to insulin hexamers.
The present work was aimed at studying the ability of three model green proteins to covalently bind to microparticles (MPs) based on poly(D,L-lactic acid) (PLA). Green fluorescent protein (sfGFP), ...the fusion protein of recombinant human β2-microglobulin (β2M) with sfGFP (β2M–sfGFP) and the fusion protein of recombinant human amylin (IAPP) with sfGFP (IAPP–sfGFP) were isolated using affinity chromatography. MP–PLAs were formed by the double-emulsion method. The modification of MP–PLAs by protein was confirmed by laser scanning microscopy (LSM). In addition, LSM was used to study the phagocytosis of MP–PLA modified by different proteins and free model proteins by macrophages. Recombinant sfGFP was shown to binds to the surface of particles at lower amounts compared to β2M–sfGFP and IAPP–sfGFP. This is probably due to the fact that protein amino groups that could potentially react with activated carboxyl groups on the surface of particles are sterically inaccessible for this reaction because of the sfGFP structure. The β2M and IAPP proteins, being components of the respective recombinant fusion proteins, are spacer structures between the surface of spherical particles and sfGFP. It was established that a threefold increase in the protein/particles ratio did not lead to an increase in the bound protein per unit of particle mass, which may indicate the amount of protein that can be bound per unit of particle mass is limited by the capacity of particles themselves. The study of phagocytosis of protein-modified MP–PLAs has shown that MP–PLAs containing model proteins (β2M–sfGFP and IAPP–sfGFP) on their surface are successfully phagocytized by macrophages and, thereby, can contribute to the activation of cell-mediated immune response, which is important for controlling various, including viral, infections. Phagocytosis of model proteins (β2M–sfGFP, IAPP–sfGFP) has also been shown in the present work. This may be due to the fact that both β2M and IAPP are amyloidogenic and aggregation-prone proteins. In all likelihood, the aggregates of these proteins can be absorbed by macrophages due to the increased size compared to their monomeric forms.
The search for two mutations, FH-Helsinki and FH-North Karelia, in LDL receptor gene was carried out in patients with familial hypercholesterolemia from St. Petersburg (80 families) and Petrozavodsk ...(80 families) using allele-specifi c PCR and analysis of single-stranded DNA fragment conformation polymorphism (SSCP analysis) with subsequent sequencing. The FH-North Karelia mutation was found in one family in St. Petersburg and in one family in Petrozavodsk, while FH-Helsinki mutation was not detected in any of the samples. Hence, the two “Finnish” mutations together responsible for 2/3 familial hypercholesterolemia cases in Finland were extremely rare in the Russian regions neighboring Finland.
Mutations in the low density lipoprotein receptor gene (
LDLR
) frequently impair folding and intracellular traffic of the receptor protein, resulting in the development of a monogenic disorder, ...familial hypercholesterolemia (FH). Identification of novel
LDLR
mutations requires confirmation of their functional importance in distinguishing pathogenic mutations from neutral changes in the aminoacid sequence. To elaborate a system for evaluation of the effect of mutation on the folding and intracellular transport of the LDLR, as well as its ability to bind low density lipoprotein (LDL), we constructed a plasmid containing
LDLR
cDNA and the gene of enhanced green fluorescent protein (EGFP). Confocal microscopy has shown that, upon transient transfection of HEK293 cells with the plasmid, the recombinant fusion protein LDLR–EGFP is transported onto the cellular membrane and binds labeled LDL. This construct will be further modified by site-directed mutagenesis to reproduce the
LDLR
missense mutations most common in the population of northwest Russia so as to study the subcellular localization and function of the modified chimeric protein.
Витамин D относится к группе жирорастворимых витаминов, который естественным образом присутствует лишь в очень ограниченном количестве продуктов питания, а синтез в организме человека возможен только ...в определенных условиях, когда ультрафиолетовые лучи солнечного света попадают на кожу. Как известно, D-гормон обладает антипролиферативным эффектом на кератиноциты, поэтому оправдано применение витамина D при пролиферативных заболеваниях кожи, например, псориазе. Цель. Изучение уровня 25(ОН) витамина D у пациентов с псориазом и псориатическим артритом в зависимости от состояния минеральной плотности костной ткани (МПКТ). Материал и методы. На базе Иркутского областного диагностического центра были обследованы 123 пациента с псориазом и псориатическим артритом (женщин 39%, мужчин 61%), средний возраст - 52,31±7,3 лет. Критериями исключения из исследования явился регулярный прием препаратов, содержащих витамин D. На всех пациентов заполнялась унифицированная анкета с детальным анализом факторов риска остеопороза. Определение уровня 25(ОН) D проводилось при помощи биохимического анализатора Cobas 8000, (Roche, Швейцария). Дефицит витамина D определяется как концентрация 25(ОН^ <20 нг/мл, недостаточность - концентрация 25(ОН^ от 20 до 30 нг/мл, адекватные уровни - более 30 нг/мл. Минеральная плотность костной ткани оценивалась методом двухэнергетической рентгеновской абсорбциометрии на аппарате EXCELL XR-46 NORLAND в области поясничного отдела позвоночника и проксимального отдела бедренной кости. Оценка плотности костной ткани осуществлялась в г/см2, по Т критерию (у лиц старше 50 лет) и Z критерию (у лиц до 50 лет) и их про центному отклонению от стандарта. Согласно рекомендациям ВОЗ (1994) снижение МПКТ по Т - критерию до -1,0 SD оценивалось как вариант нормы, от -1,0 SD до -2,5SD - остеопения, менее -2,5 SD - остеопороз. Статистическая обработка результатов проводилась с использованием пакета прикладных программ Statistica 6.0 («Statsoft», США). Результаты. Концентрация уровня 25(ОН) D в сыворотке крови у обследованных пациентов в среднем составила 19,1±1,3 нг/мл и варьировала от 7,16 нг/мл до 34,80 нг/мл. Дефицит и недостаток витамина D выявлен у 90 пациентов, что составило 73,2%. Лишь треть (26,8%) обследованных имели нормальный уровень витамина D. Установлено, что в условиях снижения концентрации 25(ОН) D в сыворотке крови показатели минеральной плотности костной ткани были существенно ниже, по сравнению с теми, у кого уровень витамина D соответствовал нормальным значениям. Так, частота остеопороза составила в группе пациентов с дефицитом и недостаточностью витамина D 53,3% (48 чел.), среди лиц с нормальным уровнем витамина - 15, 2% (р=0,0003). Средние значения ВМЭ в первой группе составили в среднем 0,706±0,06 gWmI, в второй - 0,923±0,05 g\ сш соответственно. Дефицит витамина D ассоциировался с такими факторами риска, как снижение СКФ<60 мл/мин (OR 1,9; р<0,01), склонность к падениям (OR 1,7; р<0,05), переломы в анамнезе (OR1,3; р<0,05) и ИМТ >25 кг/м2 (OR1,3; р<0,05). Выводы. Таким образом, у пациентов, страдающих псориазом и псориатическим артритом, в 70% случаев отмечался дефицит или недостаток витамина D в сыворотке крови, что ассоциировалось с низкой минеральной плотностью костной ткани и высокой частотой остеопороза.
Interaction of the dye Congo red (CR) with fibrils of three model proteins—hen egg lysozyme, recombinant human beta 2-microglobulin (b2M), and recombinant human transthyretin (TTR)—has been ...investigated using spectrophotometry. Considerable amounts of impurities were detected in the commercial dye formulation. A procedure of dye purification has been developed. The molar extinction coefficient of the dye at 490 nm (ε
490
) has been measured; the coefficient was 3.3 × 10
4
M
–1
cm
–1
at pH > 6.0. The formation of a complex between CR and the fibrils was accompanied by a change in the absorption spectrum of the dye in the visible wavelength range. Titration of fibril solutions with excessive amounts of dye showed that the number of CR molecules bound to a protein monomer within the lysozyme fibrils was close to five, whereas the respective ratio for b2M was close to four, and the ratio for TTR fibrils was close to four molecules per protein subunit.
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